U.S. patent number 7,708,908 [Application Number 12/038,138] was granted by the patent office on 2010-05-04 for carboxylic acid-modified edot for bioconjugation.
This patent grant is currently assigned to The Regents of The University of Michigan. Invention is credited to Jae Cheol Cho, Jinsang Kim, David C. Martin, Laura K. Povlich.
United States Patent |
7,708,908 |
Kim , et al. |
May 4, 2010 |
**Please see images for:
( Certificate of Correction ) ** |
Carboxylic acid-modified EDOT for bioconjugation
Abstract
An electroconductive carboxylic acid functionalized monomer
corresponding to Formula (I), wherein A represents a hydrogen or a
carboxyl group. Polymerized monomers of Formula (I) conjugated with
a biomolecule result in conjugated PEDOT polymers of Formula (III)
wherein A is a hydrogen or a carboxylic acid group and B is a
biomolecule selected from the group consisting of a peptide, a
protein, a lipid, a carbohydrate and a polynucleotide. The
biomolecule conjugated polymers can be disposed onto an
electrically conductive substrate wherein the substrate has a first
layer of PEDOT polymerized on a surface of the substrate and a
second layer of biomolecule conjugated PEDOT polymer of Formula
(III) polymerized on the first layer of PEDOT. The first and second
layers form a charge transport material in electrical communication
with the conductive substrate. The electrically conductive
substrate further comprises a dopant.
Inventors: |
Kim; Jinsang (Ann Arbor,
MI), Cho; Jae Cheol (Ann Arbor, MI), Povlich; Laura
K. (Ann Arbor, MI), Martin; David C. (Ann Arbor,
MI) |
Assignee: |
The Regents of The University of
Michigan (Ann Arbor, MI)
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Family
ID: |
39721550 |
Appl.
No.: |
12/038,138 |
Filed: |
February 27, 2008 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20080224099 A1 |
Sep 18, 2008 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60904118 |
Feb 28, 2007 |
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Current U.S.
Class: |
252/500; 549/29;
528/377; 422/79 |
Current CPC
Class: |
C07D
495/04 (20130101) |
Current International
Class: |
H01B
1/12 (20060101); C07D 333/10 (20060101); C08G
75/00 (20060101); G01N 31/00 (20060101) |
Field of
Search: |
;252/500 ;528/377
;549/29,60 ;422/79,82.02 ;600/372 ;607/115 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 2006/018643 |
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Feb 2006 |
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WO |
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WO 2008/130326 |
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Oct 2008 |
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WO |
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WO 2009/054814 |
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Apr 2009 |
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WO |
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Other References
Povlich et al "Carboxylic Acid-Modified EDOT for
Bio-Functionalization of Neutral Probe Electrodes", Polymer
Preprints (ACS) (2007), 48(1), 7-8; Spring Meeting Mar. 2007. cited
by examiner .
Mouffouk et al "Oligonucleotide-functionalized
poly(3,4-ethylenedioxythiophene)-coated microelectrodes . . . ",
Electrochem Comm 8 (2006) 317-322. cited by examiner .
Malhotra et al "Prospects of conducting polymers in biosensors",
Abalytica Chimica Acta 578 (2006) 59-74. cited by examiner .
Lee et al "Carboxylic Acid-Functionalized Conductive Polypyrrole as
a Bioactive Platform for Cell Adhesion", Biomacromolecues 2006, 7,
1692-1695. cited by examiner.
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Primary Examiner: Kopec; Mark
Attorney, Agent or Firm: Harness, Dickey & Pierce,
P.L.C.
Government Interests
STATEMENT OF GOVERNMENT RIGHTS
This invention was made with Government support under Contract No.
DMR0158079 awarded by the National Science Foundation and Contract
No. W911NF-06-1-0218 awarded by the Army Research Office. The U.S.
Government has certain rights in this invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application
No. 60/904,118 filed on Feb. 28, 2007. The disclosure of the above
application is incorporated herein by reference.
Claims
What is claimed is:
1. An electroconductive carboxylic acid functionalized monomer
corresponding to Formula (I), ##STR00014## wherein A represents a
hydrogen or a carboxyl group.
2. The electroconductive carboxylic acid functionalized monomer
according to claim 1, wherein A represents a hydrogen.
3. The electroconductive carboxylic acid functionalized monomer
according to claim 1, wherein A represents a carboxyl group.
4. The electroconductive carboxylic acid functionalized monomer
according to claim 1, further comprising a conjugated biomolecule,
wherein said carboxylic acid group of said monomer is coupled to an
amino group on said biomolecule with a carbodiimide containing
compound to form an amide bond with said biomolecule.
5. An electroconductive film or substrate coating with charge
transport properties comprising a polymer and a dopant, said
polymer comprising polymerized carboxylic acid functionalized
monomer corresponding to Formula (I): ##STR00015## wherein A
represents a hydrogen or carboxyl group.
6. An electroconductive film or substrate coating according to
claim 5, wherein said polymer is conjugated to one or more of a
peptide, a protein, a lipid, a carbohydrate or a
polynucleotide.
7. The electroconductive film or substrate coating of claim 5,
further comprising a polymer selected from the group consisting of
poly 3,4-ethylenedioxythiophene (PEDOT), polypyrrole, polyanilines,
polyacetylenes, polythiophenes, and blends thereof.
8. The electroconductive film or substrate coating of claim 5
wherein the dopant is selected from the group consisting of
poly(styrenesulfonate), phosphate-buffered saline, Hank's Balanced
Salt Solution, collagen, poly-D-Lysine, poly-L-Lysine,
poly-ornithine, dexamethasone, antibiotics, anti-mitotics, growth
factors, scar-reducing drugs, poly acrylic acid, dodecylbenzene
sulfonic acid, p-toluenesulfonic acid and combinations thereof.
9. The electroconductive film or substrate coating of claim 5,
further comprising an electrically conductive substrate wherein
said film or substrate coating is disposed on a surface of said
conductive substrate.
10. The electroconductive film or substrate coating of claim 9,
wherein said film or substrate coating is polymerized on a layer of
PEDOT, said layer of PEDOT being polymerized on a surface of said
electrically conductive substrate.
11. A biomolecule conjugated PEDOT polymer comprising a monomer of
the formula: ##STR00016## wherein A is a hydrogen or a carboxylic
acid group; and B is a first biomolecule selected from the group
consisting of a peptide, a protein, a lipid, a carbohydrate and a
polynucleotide.
12. The biomolecule conjugated PEDOT polymer according to claim 11,
wherein A is a hydrogen.
13. The biomolecule conjugated PEDOT polymer according to claim 11,
wherein A is a carboxylic acid group.
14. The biomolecule conjugated PEDOT polymer according to claim 11,
wherein B is a peptide selected from the group consisting of RGD,
GRGDS, IKVAV, CDPGYIGSR, YIGSR, KDEL and combinations thereof.
15. The biomolecule conjugated PEDOT polymer according to claim 11,
wherein B is a eukaryotic cell growth factor comprising a nerve
growth factor, an insulin-like growth factor, s-myotrophin, a
vascular smooth muscle cell growth factor, a vascular endothelial
growth factor A and a beta-transforming growth factor.
16. The biomolecule conjugated PEDOT polymer according to claim 11,
wherein B is a phosphoramidate polynucleotide having 7 to 50
nucleotides.
17. The biomolecule conjugated PEDOT polymer according to claim 11,
wherein when A is a carboxylic acid group, A is coupled to a second
biomolecule via an amide bond, and said first and second
biomolecules can be the same or different.
18. The biomolecule conjugated PEDOT polymer according to claim 11,
which is oxidatively or reductively doped to form a conducting
ionic polymer.
19. An electrically conductive substrate having a first layer of
PEDOT polymerized on a surface of said substrate and a second layer
of biomolecule conjugated PEDOT polymer of claim 11 polymerized on
said first layer of PEDOT, said first and second layers forming a
charge transport material in electrical communication with said
conductive substrate.
20. The electrically conductive substrate of claim 19, further
comprising a dopant.
Description
FIELD
The present disclosure relates to compositions comprising
carboxylic acid-modified 3,4-ethylenedioxythiophene (EDOT) and
methods of functionalizing and conjugating electrically conductive
monomers and polymers with bioactive molecules to promote cellular
interactions with the electrically conductive polymers.
BACKGROUND
The statements in this section merely provide background
information related to the present disclosure and may not
constitute prior art.
Coatings composing the charge-transporting polymer
poly(3,4-ethylenedioxythiophene) (PEDOT) have been developed for
biomedical electronic devices such as neural probes. See Cui et
al., Sensors and Actuators (2003) 89:92-102 which is hereby
incorporated in its entirety. Although the morphology and
conductivity of PEDOT make it useful as a bioelectrode coating, a
more bioactive film would be preferable.
When a neural probe is contacted with a conductive polymer for
example polythiophene or PEDOT or PEDOT derivative, inherent
problems are easily found during in vivo use. When a probe is
inserted into living tissue, for example, the brain, there is a
reactive inflammatory response, because the electrode surface lacks
the proper functionality to interact with the cells at the site of
implantation.
Therefore, in order to maintain the recording and stimulating
capabilities of neural devices, it is necessary to develop
materials that reduce the brain immune response, increase the
likelihood of establishing biocompatible connections between the
electrode and the brain cells and materials that favor the
attraction of neurons to the electrode over less favorable cell
types like glial cells.
At present, electrodes comprising of PEDOT coatings have been
designed to increase the total surface area of the electrode and
enable the electrode to interact with fine cellular processes in
order to make them more biocompatible. However, the present PEDOT
coated electrodes are not sufficiently biologically compatible with
the cells and tissues into which they are implanted.
PCT Application WO 2006/018643 describes sensors that comprise a
conjugate having a ligand attached to EDOT or derivative and
polymer thereof by means of a spacing element. This application
illustrates an example of nucleic acid coupling to EDOT via the
synthesis of an acid functionalized EDOT. The synthesis process
yields a 2,3-dihydro-thieno[3,4-b][1,4]dioxin-2-ylmethyl ester
which can be coupled to a nucleic acid using
(dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride as a
coupling agent. However, several drawbacks of using the ester or
ether forms of carboxylic acid functionalized EDOT appear when used
in a biological system. These drawbacks include enzymatic cleavage
of the conjugated polymer at the ester bond by esterases, for
example acetylcholinesterase, thus limiting the sensor or film's
ability to interface with biological tissue. Also, since the ester
form of carboxylic acid functionalized EDOT has a longer alky
chain, it is less water-soluble than the carboxylic acid described
in this application.
Further areas of applicability will become apparent from the
description provided herein. It should be understood that the
description and specific examples are intended for purposes of
illustration only and are not intended to limit the scope of the
present disclosure.
SUMMARY
The invention comprises a conductive polymer film or coating
comprising at least one layer comprising a polymer polymerized with
a monomer of Formula (I):
##STR00001##
In a further aspect, the present disclosure provides for the
synthesis and use of electroconductive carboxylic acid
functionalized monomers conjugated to one or more biomolecules,
wherein the carboxylic acid group of the monomer can be coupled to
an amino group on the biomolecule with a carbodiimide containing
compound to form an amide bond with the biomolecule.
In still a further aspect, the present disclosure provides a
biologically enhanced electroconductive polymer and methods for
using the polymer. The polymer comprises a biomolecule conjugated
PEDOT polymer of Formula (III):
##STR00002##
The polymer used to make the structure shown in Formula (III)
contains a first or a second carboxylic acid functional group. The
A shown in Formula (III) can be a hydrogen or a carboxylic acid
group and B can be a biomolecule selected from the group consisting
of a peptide, a protein, a lipid, a carbohydrate and a
polynucleotide.
In still a further aspect, the present disclosure provides an
electrically conductive substrate. The substrate can have a first
layer of PEDOT polymerized on a surface of the conductive substrate
and a second layer of biomolecule conjugated PEDOT polymer of
Formula (III) polymerized on the first layer of PEDOT. After the
first and second layers have been electropolymerized on the
substrate, the electropolymerized first and second layers form a
charge transport material in electrical communication with the
conductive substrate. The electrically conductive substrate further
comprises a counter ion or dopant in order to perform the
electropolymerization of the layers and for transferring charge
from the conductive substrate to the material comprising the first
and second layers.
DRAWINGS
The drawings described herein are for illustration purposes only
and are not intended to limit the scope of the present disclosure
in any way.
FIG. 1 depicts NMR spectra of intermediate (2) in CDCl.sub.3 as
synthesized in accordance with Scheme 1 of the present
disclosure.
FIG. 2 depicts NMR spectra of intermediate (4) in CDCl.sub.3 as
synthesized in accordance with Scheme 1 of the present
disclosure.
FIG. 3 depicts NMR spectra of intermediate (6) in CDCl.sub.3 as
synthesized in accordance with Scheme 1 of the present
disclosure.
FIG. 4 depicts NMR spectra of intermediate (7) in d.sup.6-DMSO as
synthesized in accordance with Scheme 1 of the present
disclosure.
FIG. 5 depicts NMR spectra of intermediate (8) in d.sup.6-DMSO as
synthesized in accordance with Scheme 1 of the present
disclosure.
FIG. 6 depicts a reaction scheme involving carboxylic acid EDOT
coupling using solid state coupling steps for the synthesis of
bioconjugated electrically conductive polymer in accordance with
the methods of the present disclosure.
FIG. 7 depicts a reaction scheme involving coupling of carboxylic
acid PEDOT using solution state coupling steps for the synthesis of
bioconjugated electrically conductive polymer in accordance with
the methods of the present disclosure.
FIG. 8 depicts a general scheme for making a bioconjugated PEDOT
film. First a solution of EDOT is electropolymerized as a first
layer on the surface of a conductive substrate. Then a second layer
of carboxylic acid EDOT is electropolymerized on the surface of the
first layer. The second layer of carboxylic acid PEDOT is then
conjugated to a peptide (GRGDS) by coupling the amino containing
groups of the peptide to the COOH groups of the PEDOT using
carbodiimide coupling chemistry. The resultant film is conjugated
with a biomolecule providing a biologically enhanced film for
interaction with electrically active cells like neurons.
FIG. 9 illustrates a graph showing the C 1s XPS spectra for the
PEDOT, carboxylic acid-PEDOT homopolymer film, PEDOT treated with
GRGDS peptide and the carboxylic acid PEDOT-GRGDS peptide copolymer
film. The conducting polymer films incorporated lithium perchlorate
dopant. Samples treated with GRGDS peptide were washed extensively
to remove unbound peptide.
FIG. 10 illustrates a graph showing the N 1s XPS spectra for the
PEDOT, carboxylic acid-PEDOT homopolymer film, PEDOT treated with
GRGDS peptide and the carboxylic acid PEDOT-GRGDS peptide copolymer
film. The conducting polymer films incorporated lithium perchlorate
dopant. Samples treated with GRGDS peptide were washed extensively
to remove unbound peptide.
FIG. 11 depicts an EIS spectra for a PEDOT homopolymer film, a
carboxylic acid PEDOT homopolymer film on top of a layer of PEDOT
with PSS dopant and a bare Au/Pd electrode are compared.
FIG. 12 depicts cyclic voltammetry (CV) curves of carboxylic acid
PEDOT on top of a layer of PEDOT and PEDOT coatings, all with PSS
dopant, for an average of 5 cycles. The CV curves shown in FIG. 12,
demonstrate that different behavior between the carboxylic acid
PEDOT and PEDOT coatings.
FIG. 13A is a photomicrograph of F-actin staining of C2C12 mouse
skeletal muscle cells on PEDOT-PSS. Cells were seeded for 4 hours
before fixing with formaldehyde and then staining with
phalloidin.
FIG. 13B is a photomicrograph of F-actin staining of C2C12 cells on
PEDOT-PSS-RGD. Cells were seeded for 4 hours before fixing with
formaldehyde and then staining with phalloidin.
DETAILED DESCRIPTION
The following description is merely exemplary in nature and is not
intended to limit the present disclosure, application, or uses.
The present disclosure provides a carboxylic and dicarboxylic acid
functionalized electroconductive monomer 3,4-ethylenedioxythiophene
(EDOT) according to Formula I, wherein A can be a hydrogen or a
carboxylic acid. The disclosure also provides for polymerized
carboxylic acid-PEDOT films and coatings on various solid and
flexible substrates wherein the polymerized carboxylic acid EDOT
monomer or carboxylic acid PEDOT can be further conjugated to
biomolecules via its functionalized carboxylic acid group using
standard coupling chemistries. The biomolecules can include, for
example, peptides, for example, RGD, GRGDS, IKVAV, CDPGYIGSR,
YIGSR, KDEL and combinations thereof (for example RGD-YIGSR),
proteins, nucleic acids for example, deoxyribonucleic acids (DNA)
consisting of small polynucleotide or oligonucleotide lengths of
7-50 nucleotides, nucleic acids of 50-10 kbp, ribonucleic acid
(RNA), snRNA, siRNA, miRNA, nucleic acid mimetics, PNAs and
combinations thereof), lipids, carbohydrates, including saccharides
and polysaccharides and other organic compounds having a compatible
coupling functional group to couple COOH groups. The conjugated
carboxylic acid PEDOT polymer when disposed as a film or coating on
a substrate or electrode, enhances the film or coating's
biocompatibility with cells, cellular components, tissues and other
biological samples.
In some embodiments of the present disclosure, conductive polymers
can impart desirable features. For example, they are electrically
stable over time following implantation in tissue; are relatively
non-biodegradable, yet highly biocompatible; and elicit lower
levels of immunoreactivity than commonly used conducting materials
(such as silicon, platinum, iridium, indium tin oxide, and
tungsten). As used herein, conductive polymers are conjugated
polymers that are capable of conducting electrons. The term
"conductive polymer(s)" is used interchangeably with "conducting
polymer(s)." Conductive polymers are formed from their monomeric
form (as used herein "conducting monomers") via electrochemical
polymerization, oxidative polymerization, actinic radiation
polymerization and other methods commonly used in the art.
Conducting polymer polymerized around an electrically conductive
substrate can also be referred to as a conducting polymer network
due to its three dimensional, fuzzy, soft fibrils that extend out
from the electrically conductive substrate. In some embodiments,
the conducting polymer network contains embedded biological
components including cells, cellular constituents, bioactive
molecules or substances and combinations thereof.
Synthesis of Carboxylic Acid EDOT
Scheme 1. Synthesis of
2,3-dihydrothieno[3,4-b][1,4]dioxin-2-carboxylic acid (8), herein
referred to as carboxylic acid EDOT monomer.
##STR00003## ##STR00004##
Carboxylic acid EDOT monomer can be synthesized, as shown in Scheme
1. Products formed and reacted are designated by Arabic numerals.
First thiodiglycolic acid (1, 25 g, 0.17 mol) is refluxed with 10
ml sulfuric acid in 100 ml ethanol for 12 hours. The solution is
cooled, diluted with 150 ml of water, and the product is extracted
into diethyl ether three times. The organic layer is then washed
three times with Na.sub.2CO.sub.3/H.sub.2O, dried with MgSO.sub.4
and the solvent is removed to produce 27.06 g of diethyl
thiodiglycolate (2, 79% yield). Diethyl thioglycolate (27.06 g,
0.13 mol) is then added dropwise with diethyl oxalate (50 g, 0.34
mol) to 250 ml of sodium ethoxide (0.58 mol) at 0.degree. C. After
complete addition the solution is refluxed for 1 hour to form
diethyl 3,4-dihydroxythiophene-2,5-dicarboxylate disodium salt (3).
After filtration, (3) is acidified using hydrochloric acid, and the
precipitate is filtered and washed with water. The product, diethyl
3,4-dihydroxythiophene-2,5-dicarboxylate (4), is dried and
recrystallized in methanol to produce a yield of 27.02 g (80%).
Next, (4) (8.75 g, 0.034 mol) is added to 200 ml of boiling
ethanol. Epibromohydrin (3.75 ml, 0.045 mol) and K.sub.2CO.sub.3
(0.94 g in 50 ml water) are added to the reaction. After 30
minutes, more epibromohydrin (6.5 ml, 0.079 mol) and
K.sub.2CO.sub.3 (0.5 g) are added and the solution is refluxed for
60 hours. The product is diluted with 150 ml of acidified water (5%
HCl) and extracted two times with chloroform. The organic layer is
then washed with 5% aqueous KCl, dried with MgSO.sub.4, and the
solvent is evaporated. The product, diethyl
2-(hydroxymethyl)-2,3-dihydrothieno[3,4-b][1,4]dioxin-5,7-dicarboxylate
(5) is purified by precipitation in diethyl ether. The by-product,
diethyl
2-hydroxy-2,3-dihydrothieno[3,4-b][1,4]dioxin-5,7-dicarboxylate, is
also present with (5) but this compound cannot react in the next
step so it is not separated from (5). Oxidation of the hydroxyl
group on (5) is achieved by adding a catalytic amount of pyridinium
chlorochromate (0.18 g, 0.8 mmol) and (5) (8.54 g, mixture with
by-product) to a cooled solution (0.degree. C.) of periodic acid
(15 g, 0.066 mol) in 240 ml of acetonitrile. The solution is
stirred for 3 hours as it warms to room temperature. After the
reaction, the solution is diluted with 300 ml of ethyl acetate,
washed with a 1:1 solution of brine:water, and the product is
extracted into a solution of sodium bicarbonate and water. The
aqueous later is then acidified with HCl and the product is
extracted into ethyl acetate. The organic layer is then dried with
MgSO.sub.4 and the solvent is removed. The product, diethyl
2-(carboxylic acid)-2'-dihydrothieno[3,4-b]dioxin-5,7 dicarboxylate
(6), is recrystallized in xylenes to produce a 26% yield from
compound (4) to compound (6). Compound (6) (2.8 g) is then reacted
with KOH (3 g, 0.05 mol) in 75 ml of water and 30 ml of ethanol for
1 hour at 60.degree. C. After the reaction the solvent is removed
and the product is washed with ethanol. The product is then
filtered, dissolved in 150 ml of water and acidified with HCl. The
product, diethyl 2-(carboxylic
acid)-2,3-dihydrothieno[3,4-b][1,4]dioxin-5,7-dicarboxylic acid
(7), is recovered as a white precipitate after stirring in
acidified water for 3 hours in .about.100% yield (2.36 g, 8.6
mmol). Compound (7) (2.36 g) is decarboxylated by refluxing with
copper chromite catalyst (0.24 g, 0.76 mmol) in 14 ml of freshly
distilled quinoline at 160-170.degree. C. for 2 hours. The solution
is diluted with ethyl acetate and filtered to remove catalyst. The
product is then washed with 5% HCl three times, NaCl/water twice
and extracted into 2% KOH. The aqueous layer is then acidified with
HCl and the product is extracted into ethyl acetate, dried with
MgSO.sub.4 and the solvent is removed to produce 1.17 g (75% yield)
of the final product,
2,3-dihydrothieno[3,4-b][1,4]dioxin-2-carboxylic acid i.e.
carboxylic acid EDOT (8). This reaction scheme results in a total
yield from compound 1 to compound 8 of 12%.
Products (2), (4), (6), (7) and (8) from Scheme 1 are verified
using NMR, as shown in FIGS. 1-5, along with the coupling
constants, J, shown below. Electron impact mass spectrometry was
performed on product (8), and a peak at 186.0 also confirmed the
synthesis of carboxylic acid EDOT.
##STR00005## (400 MHz, d.sup.6-DMSO) .delta. ppm (J Hz): 1.214 (t,
6H, J.sub.1,2 7.2 Hz, H.sup.1), 4.205 (q, 4H, J.sub.2,1 7.2 Hz,
H.sup.2), 10.305 (br s, 2H, H.sup.3)
##STR00006## (400 MHz, d.sup.6-DMSO) .delta. ppm (J Hz): 1.203 (t,
3H, J.sub.1,2 7.2 Hz, H.sup.1), 1.215 (t, 3H, J.sub.3,4 7.2 Hz,
H.sup.3), 4.158-4.278 (m, 4H, H.sup.2, H.sup.4), 5.281 (t, 1H,
J.sub.5,6 2.8 Hz, H.sup.5), 4.599 (dd, 1H, J.sub.6,5 2.8 Hz,
J.sub.6.6' 2.0 Hz, H.sup.6), 4.382 (dd, 1H, J.sub.6',5 2.8 Hz,
J.sub.6',6 12.0 Hz, H.sup.6')
##STR00007## (400 MHz, d.sup.6-DMSO) .delta. ppm (J Hz): 4.942 (t,
1H, J.sub.1,2 2.8 Hz, H.sup.1), 4.428 (dd, 1H, J.sub.2,1 2.8 Hz,
J.sub.2,2' 11.8 Hz, H.sup.2), 4.319 (dd, 1H, J.sub.2',1 2.8 Hz,
J.sub.2',2 11.8 Hz, H.sup.2')
##STR00008## (300 MHz, d.sup.6-DMSO) .delta. ppm (J Hz): 4.972 (t,
1H, J.sub.1,2 3.0 Hz, H.sup.1), 4.367 (dd, 1H, J.sub.2,1 3.0 Hz,
J.sub.2,2' 12.0 Hz, H.sup.2), 4.220 (dd, 1H, J.sub.2',1 3.0 Hz,
J.sub.2',2 12.0 Hz, H.sup.2'), 6.581 (d, 1H, J.sub.3.4 3.6 Hz,
H.sup.3), 6.627 (d, 1H, J.sub.4,3 3.6 Hz, H.sup.4)
Scheme 2. Synthesis of
2,3-dihydrothieno[3,4-b][1,4]dioxin-2,3-dicarboxylic acid, herein
referred to as dicarboxylic acid EDOT monomer shown as Formula
(II).
##STR00009##
##STR00010## ##STR00011##
In some embodiments, the diethyl
3,4-dihydroxythiophene-2,5-dicarboxylate (4) can be reacted with a
protected form of butane-1,2,3,4-tetraol, DIAD and TBP and then
oxidized to yield a second carboxylic acid group on the 1,4 dioxane
portion of the molecule. In such cases, the
5,7-di(ethoxycarbonyl)-2,3-dihydrothieno[3,4-b][1,4]dioxin-2,3-dicarboxyl-
ic acid intermediate can also be treated with an aqueous solution
of potassium hydroxide and then acidified with hydrochloric acid.
After filtration, the product,
2,3-dihydrothieno[3,4-b][1,4]dioxin-2,3,5,7-tetracarboxylic acid
can be decarboxylated by refluxing with copper chromite catalyst in
freshly distilled quinoline to form the final product
2,3-dihydrothieno[3,4-b][1,4]dioxin-2,3-dicarboxylic acid. When the
EDOT has been functionalized with two carboxylic acids, the
dicarboxylic acid EDOT can be further functionalized with one or
more functional groups, bifunctional groups, hetero-functional
groups which can subsequently be conjugated with one or more
bioactive molecules.
Synthesis of carboxylic acid EDOT according to Scheme 1 and
dicarboxylic acid EDOT and PEDOT according to Scheme 2 have been
developed to efficiently prepare carboxylic and dicarboxylic
acid-modified EDOT (Formulas (I) and (II)) for bioconjugation and
electrochemical polymerization. The carboxylic acid EDOT monomers
synthesized in accordance to the present disclosure can be designed
to have one or two carboxylic acid functional groups that can be
used as universal coupling groups for bioconjugation with various
biological molecules (e.g., peptides, proteins nucleic acids,
carbohydrates, lipids and combinations thereof) using commonly
known functional, bifunctional and hetero-functional coupling
chemistries. Examples of preferred covalent attachment chemistries
include amine, amide, ester, ether, and their heteroatom cognates,
e.g., sulfonamide, thioether, and so forth. Typically, each pair of
entities to be joined can jointly comprise a pair of reactive
groups, such as a nucleophile and an electrophile, one respectively
on each member of the pair.
Electropolymerization
As used herein, a carboxylic acid EDOT monomer can be polymerized
into the carboxylic acid PEDOT polymer form. In some embodiments,
the present bioconjugated films and coatings can comprise a mixture
of carboxylic acid EDOT monomers mixed with varying amounts of EDOT
to form copolymer carboxylic acid PEDOT. In some embodiments, the
carboxylic acid PEDOT having one or more free COOH moieties for
bioconjugation is a homopolymer of carboxylic acid EDOT. In some
embodiments of the present disclosure, carboxylic acid EDOT (0.01
M) can be electropolymerized in CH.sub.2Cl.sub.2 with
tetrabutylammonium perchlorate (TBAP, 0.05 M) on indium tin oxide
(ITO) electrodes and silicon wafers sputtered with Au/Pd.
Carboxylic acid EDOT and carboxylic acid EDOT/EDOT copolymer films
can be made using a 1:1 mole ratio and 0.01 M total monomer. For
comparison of electrical and chemical properties, films of EDOT
with TBAP in CH.sub.2Cl.sub.2, can also be electropolymerized. In
some embodiments, polymerizations can be galvanostatic with a
current density between 0.1-0.5 mA/cm.sup.2 and are performed for
about 5 minutes to about 50 minutes, preferably from about 10 to
about 20 minutes. The films can be washed with CH.sub.2Cl.sub.2
after polymerization and dried in air. After the carboxylic acid
EDOT or carboxylic acid EDOT/EDOT film(s) or coating(s) can be
applied to a substrate or electrode surface, the polymer films or
coatings have stable electrochemical characteristics including
reversible redox waves on cycling in either organic or aqueous
buffers. In some embodiments, EDOT is first electrochemically
polymerized with counter-ion (either PSS or lithium perchlorate) at
current density of 0.1-0.5 mA/cm.sup.2 for about 5 minutes to about
50 minutes. Carboxylic acid EDOT is then electrochemically
polymerized with counter-ion (either PSS or lithium perchlorate) on
top of the layer of PEDOT at current density of 0.1-0.5 mA/cm.sup.2
for 10 minutes. 5 to 10 minutes at the current density of 0.1
mA/cm.sup.2 may work to create a layer on top of the substrate or
electrode surface, but 10 minutes can be used to ensure a film of
carboxylic acid PEDOT has formed.
Coupling Bioactive Agents to Carboxylic Acid-PEDOT Films and
Coatings
In some embodiments, the carboxylic acid EDOT can be directly
electropolymerized onto electrically conductive solid or semi solid
surfaces, for example, glass, metal, ceramic and carbon surfaces
that are electrically conductive or contain electrically conductive
elements in contact with the surface and subsequently conjugated to
a bioactive molecule, for example a protein or peptide to form a
biocompatible electrically conductive film or coating. In some
embodiments, the carboxylic acid EDOT can be conjugated with the
biomolecule first and then electrochemically polymerized onto a
substrate or electrode in the form of a coating.
Carboxylic acid PEDOT polymer conjugated to an amino group
containing biomolecule can be synthesized as shown in Scheme 3.
##STR00012##
Carboxylic acid PEDOT can be coupled to a biomolecule to yield a
biomolecule conjugated PEDOT Formula (III), for example, a peptide,
by taking a sample of carboxylic acid PEDOT (from about 0.1 mg-to
about 500 mg) and adding 50 microliters of 5 .mu.M
1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide methiodide in MES
buffer (pH 6, MES=2-(N-morpholino)ethanesulfonic acid) per 0.3
cm.sup.2 of film surface. The mixture can be left to sit while
being agitated on a shaker plate for about 20 minutes. The
activator is then removed, and the film can be rinsed twice with
MES buffer. To this solution, 50 .mu.L of 4 mg/ml GRGDS peptide in
MES buffer can be added per 0.3 cm.sup.2 of film surface. This is
reacted for 24 hours while being agitated on a shaker plate. The
samples can be rinsed with de-ionized water and then stored in PBS.
The resultant coupled carboxylic acid PEDOT is shown in Formula
(III).
In some embodiments, biocompatible conductive coatings and films
comprising carboxylic and dicarboxylic acid EDOT compositions
according to Formulas (I) and (II) can be made by electrochemically
polymerizing the carboxylic and dicarboxylic acid EDOT monomer
compositions in the presence of LiClO.sub.4 counter ion on an
electrically conductive substrate, for example a sputtered AuPd
electrode. As shown in FIG. 6, films comprising polymerized EDOT
and carboxylic acid EDOT can be made by first electrochemically
depositing EDOT to form a first layer (a). Next, a second layer of
carboxylic acid PEDOT is deposited or layered on the PEDOT layer by
electropolymerizing carboxylic acid EDOT on the base PEDOT layer
(b). In some embodiments, the number of layers of PEDOT and the
number of layers of carboxylic acid-PEDOT can vary according to the
type of application required. Next, the carboxylic acid functional
groups on the PEDOT can be activated and then treated with a
peptide in the presence of a coupling reagent, for example, a
carbodiimide coupling reagent e.g.
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride or
1-(dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride for
coupling amino or NH.sub.2 groups to COOH functional groups on the
EDOT or PEDOT as shown in FIGS. 7 & 8. Formula (III) shows the
PEDOT polymer conjugated to a biomolecule designated B, only via
one carboxylic acid group.
##STR00013##
In some embodiments, the A group of Formula I and III can comprise
a second carboxylic group that can similarly be conjugated to a
biomolecule via amide bond formation as in Scheme 3 or
alternatively, a different coupling mechanism to yield a compatible
covalent linkage between the biomolecule and the PEDOT A carboxylic
acid functional group. In some embodiments, the B group of Formula
(III) can be a peptide, a protein, for example an antibody, a
receptor, a growth factor, for example, B can be a eukaryotic cell
growth factor, e.g. a nerve growth factor, an insulin-like growth
factor, s-myotrophin, a vascular smooth muscle cell growth factor,
a vascular endothelial growth factor A and a beta-transforming
growth factor, a lipid, a carbohydrate, for example, a saccharide
or polysaccharide, a nucleic acid (e.g. deoxyribonucleic acids
(DNA) consisting of small polynucleotide or oligonucleotide lengths
of 7-50 nucleotides, nucleic acids of 50-10 kbp, ribonucleic acid
(RNA), snRNA, siRNA, miRNA, nucleic acid mimetics, PNAs and
combinations thereof.
Methods of coupling carboxylic acid groups to various biologically
active side groups including amino groups and phosphate groups have
been described in various coupling methodologies using coupling
reagents commercially available through Pierce Inc. (Rockford, Ill.
USA).
In some embodiments, methods are provided to synthesize and
characterize RGD-functionalized PEDOT. To achieve this goal,
carboxylic acid functionalized EDOT can be synthesized to allow
conjugation between carboxylic acid EDOT and the peptide using
various coupling chemistries, including for example carbodiimide
based coupling shown in FIGS. 7 and 8. The resulting amide linkage
will be uniquely stable during the deprotection step of amino acids
used. Other commercially available or reported functionalized EDOT
can produce a linker with peptides that is unstable through the
deprotection step. In some embodiments of the present disclosure,
methods are provided for a synthetic route to make carboxylic
acid-functionalized EDOT (carboxylic acid EDOT) as shown in Scheme
1. Carboxylic acid EDOT monomer can be successfully synthesized,
characterized, and electropolymerized to form carboxylic acid PEDOT
polymer. In some embodiments, a polymer film along with a copolymer
film of carboxylic acid PEDOT and PEDOT have been partially
characterized. In some embodiments, conjugation of the peptide RGD
to both carboxylic acid EDOT and carboxylic acid PEDOT can be
performed as described in the coupling schemes shown in FIGS. 7 and
8 respectively. Subsequent characterization of the films can be
made with X-ray photoelectron spectroscopy, cyclic voltammetry,
impedance spectroscopy, and in vitro cell experiments. While other
PEDOT derivatives have been presented in literature (such as those
with sulphonate groups, biotinylated side-chains, and
oligonucleotide side-chains), the present disclosure provides for
novel biomolecule-conjugated PEDOT compositions and methods of
preparing such compositions.
In certain embodiments of the present disclosure, the conductive
polymers are functionalized with carboxylic acid and conjugated to
biomolecules with compatible coupling groups to COOH for example
amino, phosphate, thiol, sulfhydryl, thiocyanate and disulfide
groups. For example, RGD or GRGDS amino acid containing peptides
can be coupled to the carboxylic acid EDOT monomers and carboxylic
acid PEDOT polymers using reaction Scheme 3.
In some embodiments, the conducting polymers can include, but are
not limited to: poly(3,4-ethylenedioxythiophene) (PEDOT),
polythiophenes, polymer blends thereof, and composites with the
ability to conduct electronic charge or ions, and hybrid
polymer-metal materials that are electrically or ionically
conductive. Other conductive polymers can include functionalized
copolymers that are made from EDOT and other conducting polymers
that are functionalized with carboxylic acid and conjugated to
biomolecules, including peptides and proteins for example, RGD,
IKVAV, YIGSR, KDEL peptides and combinations thereof, for example
RGD and YIGSR, and other biomolecules containing chemically
compatible functional groups that can be covalently attached using
standard coupling chemistries to the functionalized conducting
monomer, for example carboxylic acid-EDOT. A covalent attachment
can be effected using any covalent chemistry described herein.
Typically, each pair of entities to be joined can jointly comprise
a pair of reactive groups (such as a nucleophile and an
electrophile), one respectively on each member of the pair, as
shown in the coupling reactions in FIGS. 7 and 8. Where the
biological entity (tissue, cell, cell fragment, organelle, or other
biologic) is to be directly attached to the monomer or polymer,
each will contain one reactive group of a pair. In some
embodiments, the biomolecule can be covalently attached to a
linker. Where attachment is to take place through a linker, the
linker can contain two reactive groups, one of which is capable of
covalently reacting with a reactive group of the carboxylic acid
EDOT monomer and the other of which is capable of covalently
reacting with a reactive group of the biological entity. The
reactive group(s), such as carboxylic acid, can be already present
as part of the monomer (e.g., carboxylic acid EDOT). Where
attachment is to take place through a linker, the linker can be
attached first to the polymer, first to the biological entity, or
concurrently to both. Non-limiting examples of preferred
nucleophile and electrophile groups for use in forming a covalent
attachment are presented in Table 1.
TABLE-US-00001 TABLE 1 Exemplary Reactive Group Pairs For
Attachment Chemistries between the biomolecule and the functional
group on the EDOT monomer or PEDOT polymer Nucleophile Electrophile
Attachment Amine Alkyl carbodiimide-activated ester Amide
Bromoacetamide Amine Carboxyl Amide Chloroacetamide Amine Cyclic
carboxylic anhydride Amide 9-Fluorenylmethoxycarbonyl Amide
N-Hydroxysuccinimide ester Amide Isocyanate Urea Isothiocyanate
Thiourea Phosphate Phosphoramide Phosphonate Phosphonamide
In some embodiments, conducting polymers can be any non-conductive
monomer or polymer that can be made conductive in the presence of
an appropriate doping system. In some embodiments, conjugated
polymers described herein are functionalized by chemically
synthesizing the electrically conductive monomer or polymer to
contain functional side groups (e.g., carboxylic acid) that can
allow for binding of peptides, proteins, lipids, carbohydrates and
nucleic acids before or after polymerization. In some embodiments,
the conductive polymer can be biodegradable and will dissolve in
the presence of biological fluid, for example, when the device is
implanted in situ (e.g., implantable brain prostheses, neural
stimulators, transient heart devices, and the like. The
biodegradable conducting polymer can include, for example,
polypyrrole and poly(3,4-ethylenedioxythiophene) block PEG, and
poly(3,4-ethylenedioxythiophene) PEDOT, tetramethacrylate and
others that are commercially available from TDA Research Inc.,
Wheat Ridge, Colo., USA.
Conductive carboxylic acid monomers for example EDOT, contemplated
by the present disclosure typically require counter ions for
polymerization and electroconductivity across the electrode-tissue
interface. The conducting polymers are reacted with a
polyelectrolyte at the molecular level. Electron delocalization is
a consequence of the presence of conjugated double bonds in the
conducting polymer backbone. To make the conducting polymers
electrically conductive, it is necessary to introduce mobile
carriers into the double bonds, which is achieved by oxidation or
reduction reactions (called "doping"). The concept of doping
distinguishes conducting polymers from all other kinds of polymers.
This process can be assigned as p-doping or n-doping in relation to
the positive or negative sign of the injected charge in the polymer
chain by analogy to doping in inorganic semiconductors. These
charges remain delocalized, being neutralized by the incorporation
of counter-ions (anions or cations) denominated dopants. Suitable
dopants and methods of doping are known to those skilled in the
art, e.g. from EP 0 528,662 and U.S. Pat. No. 5,198,153 or WO
96/21659. In certain embodiments, ionic electrolytes or dopants
used to polymerize conducting polymers include, but are not limited
to: poly(styrene sulfonate) (PSS; Sigma Aldrich, St. Louis, Mo.,
USA), LiClO.sub.4, Phosphate-buffered saline (PBS; HyClone, Logan,
Utah, USA), Hank's Balanced Salt Solution (HBSS, HyClone),
Collagen, Poly-D-Lysine (PDL), Poly-L-Lysine, poly-ornithine, and
bioactive molecules of interest having the appropriate ionic charge
for the type of doping system used and can include, but is not
limited to: dexamethasone or other anti-inflammatory agents,
antibiotics, anti-mitotics, growth factors, scar-reducing drugs,
poly acrylic acid, dodecylbenzene sulfonic acid (DBSA),
p-toluenesulfonic acid (p-TSA) and combinations thereof. When
electrons are used as carriers, suitable dopants are for example
halogens (e.g. I.sub.2, Cl.sub.2, Br.sub.2, ICl, ICl.sub.3, IBr and
IF), Lewis acids (e.g. PF.sub.5, AsF.sub.5, SbF.sub.5, BF.sub.3,
BCl.sub.3, SbCl.sub.5, BBr.sub.3 and SO.sub.3), protonic acids,
organic acids, or amino acids (e.g. HF, HCl, HNO.sub.3,
H.sub.2SO.sub.4, HClO.sub.4, FSO.sub.3H and ClSO.sub.3H),
transition metal compounds (e.g. FeCl.sub.3, FeOCl,
Fe(ClO.sub.4).sub.3, Fe(4-CH.sub.3C.sub.6--H.sub.4SO.sub.3).sub.3,
TiCl.sub.4, ZrCl.sub.4, HfCl.sub.4, NbF.sub.5, NbCl.sub.5,
TaCl.sub.5, MoF.sub.5, MoCl.sub.5, WF.sub.5, WCl.sub.6, UF.sub.6
and LnCl.sub.3 (wherein Ln is a lanthanoid), anions (e.g. Cl.sup.-,
Br.sup.-, I.sup.-, I.sub.3.sup.-, HSO.sub.4.sup.-, SO.sub.4.sup.2-,
NO.sup.3-, ClO.sub.4.sup.3-, BF.sub.4.sup.-, PF.sub.6.sup.-,
AsF.sub.6.sup.-, SbF.sub.6.sup.-, FeCl.sub.4.sup.-,
Fe(CN).sub.6.sup.3-), and anions of various sulfonic acids, such as
aryl-SO.sub.3.sup.-). When holes are used as carriers, examples of
dopants are cations (e.g. H.sup.+, Li.sup.+, Na.sup.+, K.sup.+,
Rb.sup.+ and Cs.sup.+), alkali metals (e.g., Li, Na, K, Rb, and
Cs), alkaline-earth metals (e.g., Ca, Sr, and Br), O.sup.2,
XeOF.sub.4, (NO.sub.2.sup.+) (SbF.sub.6.sup.-), (NO.sub.2.sup.+)
(SbCl.sub.6.sup.-), AgClO.sub.4, H.sub.2 IrCl.sub.6,
La(NO.sub.3).sub.36H.sub.2O, FSO.sub.2OOSO.sub.2F, Eu,
acetylcholine, R.sub.4N+, (R is an alkyl group), R.sub.4P.sup.+ (R
is an alkyl group), R.sub.6As.sup.+ (R is an alkyl group), and
R.sub.3S.sup.+ (R is an alkyl group).
Characterization
The electrodes, electrode-based devices, films and coatings used to
modify preexisting electrodes can optionally include controllers,
analyzers and other sensing devices and computers that can be used
to control the output of electrical current, or voltage. These
optional components can also be used to perform, measure and record
electrical events, current flow, electrical impedance spectroscopy,
cyclic voltammetry, resistance, conductance, capacitance, and
potential of the integrated network to the flow of electrons. These
analytical systems and devices are commercially available (e.g.,
the Brinkman's (Eco Chemie) Autolab system connected to various
CPU's (Windows or Macintosh computers) available from Brinkman
Instruments Inc., Westbury, N.Y., USA).
The appearance of the carboxylic acid PEDOT, PEDOT-acid/PEDOT
copolymer, PEDOT films and carboxylic acid-biomolecule conjugated
PEDOT can be characterized using optical microscopy. The chemical
compositions can be investigated using X-ray photoelectron
spectroscopy (XPS). Cyclic voltammetry (CV) from 0.5 to -0.9 V
along with electrochemical impedance spectroscopy (EIS) can be used
to characterize the electrical properties of the films.
In some embodiments, the PEDOT-acid, PEDOT-acid/PEDOT copolymer,
and PEDOT films can form bluish-green films on both ITO and Au/Pd
electrodes. The polymer formed a well-adhered film on the Au/Pd
electrode, but can delaminate off the ITO electrodes after
polymerization. As shown in FIGS. 9 and 10, the XPS
characterization was performed using the Kratos Axis Ultra XPS in
EMAL (http://www.emal.engin.umich.edu/instruments/xps.html) with
the monochromatic Al x-ray source. The spectra were all shifted so
that the C--C peak is at 285 eV.
FIG. 9 illustrates a graph showing the C 1s XPS spectra for the
PEDOT, carboxylic acid-PEDOT film on top of a layer of PEDOT, PEDOT
treated with GRGDS peptide and the carboxylic acid PEDOT-GRGDS
peptide copolymer film. The C 1s XPS spectrum in FIG. 9 supports
that the carboxylic acid functionalized EDOT has polymerized on the
top of the PEDOT film.
FIG. 10 depicts N 1s XPS spectra demonstrating that the peptide
coupling process appears to be successful. There are 2 peaks for
the peptide-treated sample, which are from the amide peptide
backbone and from the side chain in the amino acid arginine (R).
The control samples do have a nitrogen peak and we are currently
investigating the source of this nitrogen. It is predicted that the
contamination is due to impurities introduced during the
electrochemical polymerization process.
As shown in FIG. 11, the Electrochemical Impedance Spectroscopy
(EIS) spectra for a PEDOT homopolymer film, a carboxylic acid PEDOT
film on top of a layer of PEDOT (both with PSS dopant) and a bare
Au/Pd electrode are compared. The electrodes used to measure the
impedance of the two films prepared in accordance with FIG. 6 and
bare electrode were 6 mm barbell AuPd electrodes. The measurements
were taken using an Autolab potentiost/galvanostat using a
3-electrode cell (working, counter and Ag/AgCl reference
electrode). The electrodes were immersed in a PBS/water
electrolyte. Impedance measurements were taken using Frequency
Response Analyze version 4.9.005 software and the cyclic
voltammetry (CV) measurements were taken using General Purpose
Electrochemical System version 4.9.005 software. Both the
impedances of the carboxylic acid PEDOT and PEDOT have similar
electrical properties. Both films decrease the impedance of the
electrode at all frequencies, which is an important property for
biological interface applications.
FIG. 12 shows the CV curves as an average of 5 cycles. The CV
curves shown in FIG. 12 demonstrate slightly different behavior
between the carboxylic acid PEDOT on top of a layer of PEDOT and
PEDOT coatings, since the carboxylic acid PEDOT film prepared in
accordance to FIG. 6 has a higher charge capacity. Both polymer
films demonstrate some charge capacity, especially compared to the
bare Au/Pd electrode.
In some embodiments, both solid-state and solution coupling methods
can be used and are shown in FIGS. 7 and 8 respectively, for films
in which the carboxylic acid EDOT is conjugated to the peptide
before electropolymerization and for films produced where the
carboxylic acid EDOT is electropolymerized into carboxylic acid
PEDOT first, then the carboxylic acid moieties in the polymer are
coupled with RGD peptides. For solution-state coupling, one end of
the peptide can be attached to a polystyrene bead with a weak
covalent linkage. The carboxylic acid EDOT can be coupled with the
free end of the peptide to form an amide bond and after coupling,
the link between the polystyrene bead and the peptide will be
cleaved to yield a free carboxylic acid. Electropolymerization can
be performed after the coupling reaction to form RGD-functionalized
PEDOT. For solid-state coupling, electropolymerization can be
performed first in order to form a carboxylic acid PEDOT
homopolymer film on the substrate, or on a substrate first coated
with PEDOT or other conjugated electroconductive polymer as shown
in FIG. 6 with PEDOT. Before coupling, the peptide can be cleaved
from the polystyrene bead and, in order to prevent unwanted
reactions, a protecting group can be added to the carboxylic acid
end of the peptide. After the protecting group is added, the
peptide can be coupled with carboxylic acid PEDOT to form a
biomolecule conjugated electrically conductive polymer. The
protecting group will then be removed; resulting in the formation
of RGD-conjugated PEDOT film or coating. In some cases protecting
groups on the peptide are not necessary and the peptide can be
coupled without interference from functional groups. This allows
the peptide coupling to be performed in water and also eliminates
damage caused to the polymer film by the harsh deprotection
reaction conditions.
In some embodiments, RGD-conjugated PEDOT can be characterized
using XPS, EIS, CV, and, most importantly, cell experiments. In
some embodiments, the RGD-conjugated carboxylic acid EDOT is
water-soluble and can be polymerized around living cells, to form
biologically integrated bioelectrode devices comprising a first
electrically conductive substrate, a biological component (such as
a tissue cell, cell membrane or synthetic cell or micelle), and a
conductive polymer film or coating conjugated with a biomolecule.
The conductive polymer (i.e. a film or coating on an electrode
consisting of a biomolecule coupled to PEDOT) couples the
conductive substrate (e.g., an electrode) to the cells or tissue to
collectively define a bioelectrode. In some embodiments, the
bioelectrode is capable to transmit or receive an electrical signal
between the electrode and either or both of the cells or tissues
and conductive polymer.
Applications for Bioconjugated Electroconductive Films
In some embodiments, films and coatings comprising the carboxylic
acid functionalized EDOT monomer as shown in Formula I can serve as
an enhanced substrate for binding of other electroconductive
polymers. In some embodiments, the acid-EDOT of the present
disclosure can be electropolymerized onto a variety of surfaces
including metallic surfaces including, for example, gold, silver,
platinum, iridium, indium tin oxide, titanium and tungsten. In some
embodiments, other functional groups can be attached to the free
carboxylic acid moiety on the conjugated PEDOT films and coatings
which enable the functionalized conjugated PEDOT films to bind to
difficult to bind metal surfaces such as stainless steel. In this
sense, the acid-PEDOT films and coatings of the present disclosure
can act as adhesion promoters for other materials, including PEDOT
and other electroconductive conjugated polymers. As illustrative
examples, functionalized PEDOT films of the present disclosure can
then serve as a substrate for subsequent binding of other
conjugated electroconductive polymers including PEDOT, polypyrrole,
polyaniline, polyacetylenes, polythiophenes and blends thereof.
Limitations associated with electropolymerization of conducting
polymers in biological tissues can include problems with the focal
adhesion of neural cells after polymerizing EDOT directly around
living cells. PEDOT when polymerized around cells can grow on top
of the extracellular matrix (ECM), thus preventing the cells from
creating focal contacts with the ECM proteins. The lack of adhesion
can be demonstrated by the loss of actin stress fibers in the cells
and eventually cell death occurs. Since the peptide sequence RGD is
known to promote cell adhesion to the ECM, the functionalization of
electrically conductive PEDOT polymer with RGD should promote the
formation of focal contacts between neural cells and the PEDOT
film. Therefore, after polymerization of the PEDOT around living
cells, the actin stress fibers should remain intact, which will
make long-term cell survival more probable.
EXAMPLES
Example 1
Biologically Compatible Probes
An electrochemical cell probe was produced to determine whether a
biomolecule functionalized PEDOT film could be used to enhance the
compatibility between the electrode and the mouse skeletal muscle
cell line C2C12 cells. The biomolecule functionalized PEDOT film
was produced in accordance to the PEDOT/carboxylic acid PEDOT
layered film described in FIG. 6. The substrate was coated with a
layer of PEDOT followed by a plurality of layers of carboxylic acid
PEDOT doped with poly(styrenesulfonate)(PSS) in accordance with the
present disclosure. To the carboxylic acid PEDOT present on the
substrate, RGD was coupled to the available COOH groups on the
carboxylic acid PEDOT using
1-(dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride. The
resultant conjugated RGD-PEDOT/PSS substrate was subsequently
rinsed in physiological buffer and prepared for cell culture of
C2C12 cells. C2C12 cells were seeded at a density of
1.times.10.sup.5 cells per well in 6 well cell culture plates
without serum. The conjugated PEDOT/PSS was compared to conjugated
RGD-PEDOT/PSS by adding the mouse skeletal muscle cell line C2C12
onto the two substrates to determine which substrate provided a
more biocompatible surface for attachment and growth of the C2C12
cells in vitro. Cells were seeded for 4 hours before fixing with
formaldehyde and then staining with phalloidin.
As shown in FIGS. 13A and 13B, F-actin staining of C2C12 cells on
PEDOT-PSS (FIG. 13A) and PEDOT-PSS-RGD (FIG. 13B) reveals that most
C2C12 cells on PEDOT-PSS have a very round morphology whereas the
cells on PEDOT-PSS-RGD have long extensions, indicating attachment.
These results suggest that electrically active cells, for example,
C2C12 cells are more likely to attach and grow on PEDOT-PSS-RGD
containing substrates and coatings than PEDOT alone. The enhanced
compatibility of biomolecule conjugated PEDOT according to the
present disclosure can be individually manipulated for a particular
cell or tissue type by matching a peptide, growth factor, cytokine,
cluster of differentiation marker (CD) marker that can attract the
cell type or induce growth and extension of the cell on the
biomolecule conjugated PEDOT substrate.
In some embodiments, the activity, growth and differentiation of
various electrically active cells including skeletal muscle cells,
cardiac muscle cells for example cardiomyocytes and brain tissue
cells, for example neurons can be sensed by growth of these cells
on the films and coatings comprising the biomolecule conjugated
PEDOT films and coatings of the present disclosure.
Neural probes capable of electrical sensing and recording using
PEDOT are superior to bare electrodes when implanted into the
brain. These PEDOT probes however, have some limitations, including
growth of the PEDOT on top of the extracellular matrix (ECM), thus
preventing the cells from creating focal contacts with the ECM
proteins. The lack of adhesion is demonstrated by the loss of actin
stress fibers in the cells and eventually cell death occurs. In
some embodiments, a neurological sensing probe or probe array
commonly used for recording electrical activity in various portions
of the brain can be conjugated with a biomolecule for example an
RGD peptide or growth factor for example nerve growth factor in
accordance with the functionalizing and conjugation methods of the
present disclosure, to render the electrode or array more
biologically compatible with the neural cells in contact with the
probe or array of probes.
In some embodiments, the present biomolecule conjugated conductive
substrates comprising biomolecule conjugated PEDOT (Formula III)
can be used to identify and screen for biomolecules such as organic
molecules, peptides, proteins, carbohydrates and lipid molecules
capable of enhancing growth or inhibiting cell death when incubated
with an electrically active cell. As an illustrative example,
collateral sprouting of axons from the peripheral nervous system
(PNS) into the central nervous system (CNS) appears to involve the
action of a growth factor with properties similar to NGF. The
identification of specific molecules such as those found in small
molecule libraries, combinatorial libraries or peptide libraries
(Commercially available from GenScript, Piscataway, N.J. USA). The
peptides or other small molecules can be functionalized by adding a
compatible functional group, for example an NH.sub.2 or amino group
that can be conjugated to COOH functional group on the PEDOT. Other
coupling strategies commonly known can be employed in establishing
a direct coupling between the COOH group of the PEDOT polymer and
the corresponding functional group on the candidate molecule. The
library can be disposed on a substrate for example a metallic,
silicon or glass slide in single or an array pattern. The substrate
is preferably electrically conductive and can accommodate the
electropolymerization of carboxylic acid EDOT to carboxylic acid
PEDOT on the substrate. The COOH groups of the carboxylic acid
PEDOT can then be conjugated with a compatible functional group on
the peptide or small molecule in the library. The degree of
attachment, growth or differentiation of electrically active cells,
for example, neurons to the individual spots on the array can be
electrically determined by measuring the shift in redox potential
and/or capacitive charging element in the cyclic voltammogram (as
shown in FIG. 13 herein). Alternatively, the substrate containing
the peptide library or small molecule library incubated with cells
can be stained with an antibody that is capable of measuring a cell
skeletal protein, such as actin, to indicate differentiation and
growth of the cells. Identification of candidate molecules that are
capable of affecting neuronal growth should lead to an
understanding of the etiology of degenerative neurological diseases
such as Alzheimer's disease and, hopefully, to rational therapeutic
approaches.
Example 2
Biomolecule Sensing Chip
In some embodiments, the present disclosure provides for films and
coatings that are capable of sensing specific binding events
between two biomolecules, for example, an enzyme and its cognate
ligand, or a single strand of a polynucleotide and its
complementary binding polynucleotide. In some embodiments, an
electrically conductive substrate is provided that has been
electropolymerized with a layer of PEDOT or carboxylic acid PEDOT.
To the first layer, a second layer or region (which can include one
or more spots or an array of spots) of conjugated electroconductive
polymer such as carboxylic acid PEDOT, PEDOT, polyaniline,
polypyrrole, polythiophene or combinations thereof is
electropolymerized. The second layer or region can be
functionalized with carboxylic acid or carboxylic acid and any
further functional groups commonly known to react with COOH to form
a new and different functional group in accordance with the present
disclosure or methods of functionalizing COOH groups known in the
art. To the substrate a biomolecule functionalized with a
compatible functional group capable of attaching covalently to
either a COOH group or a different functional group, for example
amine, amide, hydroxyl, thiol, haloacyl or haloacetyl and SH. The
nucleophilic group of the modifying compound is selected from amine
group, a hydroxyl group, a thiol group, hydrazide and a guanidino
group.
In some embodiments, the biomolecule can be attached to a
bifunctional linker selected from the group of bifunctional linkers
having a nucleophilic group or a combination of such bifunctional
linkers. In some embodiments, the functional group can be any
nucleophilic group from an amine group, a hydroxyl group, a thiol
group, a guanidine group and hydrazide. Suitable bifunctional
linkers are well known in the art and can be found, for example, in
the catalog of the Pierce Company, Rockford, Ill. USA (Pierce
2005-2006 Applications Handbook & Catalog at
www.piercenet.com).
In some embodiments, the biomolecule can be any one or more of a
peptide, protein or a polynucleotide and combinations thereof. The
biomolecule can be treated to ensure that free primary amino groups
are available for conjugation with the substrate layered carboxyl
acid PEDOT. Naturally, proteins have one or more primary amino
groups. Oligonucleotides can also be conjugated to primary
amine-containing molecules by modifying the 5' phosphate group of
oligonucleotides using the carbodiimide crosslinker EDC and
imidazole and amine-modification of the oligonucleotide with an
excess of ethylenediamine as described in TECH TIP #30 "Modify and
label oligonucleotide 5' phosphate groups." Pierce Company,
Rockford Ill. USA. The anchoring of the biomolecule onto the
conductive substrate can be verified by reflectance infrared
spectroscopy or changes in electrochemistry of the conjugated
COOH-PEDOT.
The carboxylic acid PEDOT film and substrate coatings of the
present disclosure can be coupled to a variety of biomolecules
described above for the detection and quantification of target
ligands and complementary polynucleotides for the detection of DNA
or RNA in a test sample. With reference to DNA and RNA molecules
the nucleic acids can first be functionalized to add a primary
amino group first to generate for example, oligonucleotides can be
incubated with a carbodiimide crosslinker e.g. EDC (Pierce Co.,
Rockford, Ill. USA). The oligonucleotide as an ester intermediate
is then incubated with imidazole to yield a reactive
phosphorylimidazolide. The phosphorylimidazolide is then incubated
with excess ethylenediamine to produce a phosphoramidate
oligonucleotide that can be conjugated to a carboxylic or
dicarboxylic acid EDOT of Formula I or II or alternatively to
carboxylic acid PEDOT (Formula III). In some embodiments, the
oligonucleotide can be synthesized using automated oligonucleotide
synthesis as phosphoramidate oligonucleotides commercially
available from Operon Biotechnologies Inc., Huntsville, Ala. USA.
The conjugated PEDOT-oligonucleotides can be deposited onto an
electrically conductive substrate, for example, silica, metal or
glass substrates with electrically conductive elements and reacted
with a test nucleic sample or multiple nucleic acid samples. The
deposition process can involve any commonly known patterning
deposition technique, for example, ink jet printing, multi-pipette
deposition and the like. Upon binding of the conjugated biomolecule
to its cognate ligand or complementary DNA or RNA sequence, a
change in the electrochemical properties of the film and coatings
can be detected using electrical impedance spectroscopy XPS binding
plots and cyclic voltammetry thus illustrating their applicability
as films and coatings for Protein/peptide and DNA/RNA chips.
The description of the invention is merely exemplary in nature and,
thus, variations that do not depart from the gist of the invention
are intended to be within the scope of the invention. Such
variations are not to be regarded as a departure from the spirit
and scope of the invention.
* * * * *
References